Affinity separations utilize the natural affinity that one molecule has for another molecule to effect a separation. Bioaffinity separations are defined as affinity separations in which one of the components is of biological interest or has biological activity. Thus, bioaffinity separations involve at least one biomacromolecule, such as a protein or nucleic acid, as one of the components of the binding pair. Examples of bioaffinity binding pairs include, but are not limited to, the following: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid, reactive dye-protein, reactive dye-nucleic acid, biotin-avidin and Protein A--IgG.
Bioaffinity separations are conducted on bioaffinity selective adsorbent to which a member of the bioaffinity binding pair has been attached either covalently or by non-specific adsorption. The other member of the binding pair then binds to its counterpart when it is placed in contact with this bioaffinity selective adsorbent. Once the members of the bioaffinity binding pair have bound each other, then the member which is not placed in contact with the bioaffinity selective adsorbent can be eluted with a wide number of reagents, depending upon the experimental parameters.
Weetal, Methods in Enzymology, Volume XLIV; Immobilized Enzymes, Chapter 10, 134, Ed. K. Mosbach, Academic Press, NY (1976), describes various approaches by which to covalently modify inorganic carrier surfaces. Although covalent attachment of a member of a bioaffinity binding pair is preferred, it has some disadvantages, such as, increased nonspecific binding of undesirable proteins to the immobilized molecule, alteration of the binding properties of the covalently attached molecule and inability to recover scarce or expensive molecules because the molecule is irreversibly bound to the carrier.
Messing, Methods in Enzymology, Volume XLIV: Immobilized Enzymes, Chapter 11, 149, Ed. K. Mosbach, Academic Press, NY (1976), sets forth another commonly used attachment technique, nonspecific adsorption of a member of a bioaffinity binding pair to the carrier. This technique also has disadvantages such as the inability to retain the nonspecifically adsorbed molecule on the carrier due to the weak forces binding the molecule to the support. Another problem is partial alteration of the binding properties of the molecule adsorbed to the carrier.
Other approaches have been developed in the art for immobilizing biological molecules to a plastic surface by physically or chemically derivatizing the surfaces.
U.S. Pat. No. 4,657,873 issued to Gadow et al. on Apr. 14, 1987, discloses preactivation of plastic surfaces by coating the surface with an amphoteric polypeptide consisting of phenylalanine and lysine amino acids. The hydrophobic phenylalanine residues enable the polypeptide to adsorb to the plastic surface and the hydrophilic lysine residues are available for immobilizing organo-chemical and biological materials. One problem with the Gadow approach is that the polypeptide of interest is not readily available and is very expensive to obtain. In addition, polypeptides may be susceptible to attack by proteases which may be present in clinical samples. Whether proteases will degrade a polypeptide coated on a plastic surface depends on the tertiary structure of the polypeptide on the surface. If inadvertent digestion of the polypeptide occurred, then a large error would be introduced into assay results.
Similarly, Gadow et al., J. Clin. Chem. Clin. Biochem., pages 789-797, vol. 21, no. 12 (1983), discloses the adsorption of poly phe-lys to polystyrene balls followed by activation with glutaraldehyde and coupling of the required ligand. This technique has the same deficiencies as the technique discussed above in connection with U.S. Pat. No. 4,657,873.
U.S. Pat. No. 4,654,299 issued to Lentfer on Mar. 31, 1987, discloses immobilizing proteins on polystyrene surfaces which have been preactivated with a bis-diazonium compound. The major drawback of this procedure is that bis-diazonium compounds and their precursors, benzidines, are highly carcinogenic.
U.S. Pat. No. 4,444,879, issued to Foster et al. on Apr. 24, 1984, discloses a solid-phase support for immobilizing reactants of an immunoreaction which comprises a light-transparent, water-insoluble article which is inert to chemical reaction with the reactants and a dried film of a synthetic, polymeric resin having chemical groups attached which are capable of forming covalent bonds with the reactants. At least a portion of the article is coated with the dried film.
U.S. Pat. No. 4,001,583, issued to Barrett on Jan. 4, 1977, discloses pretreating plastic surfaces with glutaraldehyde to covalently bind biological substances to the plastic surface. Glutaraldehyde is polymerized directly on the inside surface of a plastic material with or without previously treating the surface with an aliphatic amine or diamine.
U.S. Pat. No. 4,279,787, issued to Huizinga on July 21, 1981, discloses a method of binding antigenically active materials to the surface of water insoluble hydrophobic polymeric substances which comprises pretreatment with an aldehyde of the formula, OHC-X-CHO wherein X may be an alkylene or cycloalkyl group.
U.S. Pat. No. 4,352,884, issued to Nakashima et al. on Oct. 5, 1982, discloses a carrier coated with a hydrophilic acrylate copolymer for immobilizing bioactive materials.
U.S. Pat. No. 4,278,651, issued to Hales on July 14, 1981, discloses a solid support having a water insoluble polymer including at least one functional group through which a receptor is covalently linked to the support.
European patent application No. 83109418.0 discloses plasma modification techniques to chemically modify polymeric surfaces in order to attach large molecules.